The alpha-crystallins make up a large fraction of the soluble protein in the vertebrate lens, where they are believed to function as structural proteins, are the first crystallins to be expressed in the developing mouse lens, and are a relatively small family of crystallins encoded by only two genes, the alphaA- and alphaB-crystallin genes. The alpha-crystallins are related to the small heat shock proteins, exhibit molecular chaperon activity, and kinase activity, and, at least in the case of alphaB-crystallin, have been shown to be expressed in a variety of nonlenticular tissues where their function is unknown. Toward understanding the role of the alpha-crystallins in lens and nonlens tissues, we are functionally deleting alpha-crystallins by disrupting the alpha-crystallin genes in mice. We are employing the technique of homologous recombination in pluripotent mouse embryonic stem cells followed by generation of chimeric mice containing the altered stem cells. Using genomic clones of the alphaA- and alphaB-crystallin genes and the transcription factor alphaA-CRYBP1 gene, which we previously isolated from a mouse 129SV library (the same strain as most of the embryonic stem cell lines currently in use), we have constructed alphaA-crystallin, alphaB-crystallin and alphA- CRYBP1 gene knockout vectors. Several embryonic stem (ES) cell clones containing gene knockouts have been isolated for each of the three genes. Lines of mice have been generated from two alphaA-crystallin gene knockout ES cell clones and from four alphaB-crystallin gene knockout ES cell clones. Several chimeric founder mice have been generated from two lines of alphaA-CRYBP1 gene knockout ES cell clones. In collaboration with Nicholas Ambulos (University of Maryland Medical School), we have sequenced 15 kb of the mouse alphaA-crystallin gene locus.